Expression of Human Therapeutic Proteins in Chloroplasts

描述（由申请人提供）：干扰素（IFN）是细胞因子， 抗病毒、抗生长和免疫调节特性。IFNalpha 2被使用 作为白血病、转移癌、卡波西肉瘤和病毒性 肝炎IFN α 2治疗的年费用为4000美元， 不适用于全球大多数患者。在1.2亿感染者中， 丙型肝炎病毒，70%有肝功能异常，33%有 严重肝硬化在肝脏中表达的IFN α 5具有最有效的抗病毒作用 活性，使其成为一种有吸引力的治疗候选物。胰岛素样生长 因子I（IGF-I）是一种有效的多功能合成代谢激素， 肝，它的缺乏导致几个系统性并发症发生 在肝硬化中。每例糖尿病患者每年对IGF-1的需求量为 600毫克，每毫克的成本是30000美元。 转基因叶绿体技术为基因工程提供了一种新的解决途径 由于超表达能力和折叠能力， 并处理具有二硫键的真核蛋白质（从而消除 需要昂贵的纯化后处理）。烟草是理想的选择 由于其生物量大，易于扩大规模（百万粒种子笔植物）， 基因操纵和迫切需要探索这种替代用途 有害作物功能性生物药物的口服给药减少了90 百分之生产成本，消除了昂贵的 洁净.此外，植物细胞内的生物封装提供了 在消化过程中防止蛋白水解降解。给予干扰素 口服对人和动物具有生物活性。 因此，IFN α 2、IFN α 5和IGF-1将表达如下： 开发用于增强表达或口服递送的重组DNA载体B） 产生转基因烟草和番茄植物 使用分子和生物化学方法表达蛋白质或融合蛋白 d）从转基因叶绿体中纯化治疗性蛋白质 表征和比较治疗性蛋白质（产量、纯度、功能性） f）体外和体内研究 （临床前试验）蛋白质生物功能性，肠胃外和口服递送。 通过转基因叶绿体的大规模和低成本生产应该提供 以负担得起的费用为患者提供治疗，烟农可替代用途 这种危险作物的种植

DESCRIPTION (provided by the applicant): Interferons (IFN) are cytokines with antiviral, antigrowth and immuno-modulatory properties. IFNalpha 2 is employed as a therapy for leukemia, metastasizing carcinoma, kaposi sarcoma and viral hepatitis. The annual cost of IFNalpha 2 therapy is $4000, making it unavailable for most patients worldwide. Among 120 million people infected with hepatitis C virus, 70 percent have abnormal liver function and 33 percent have severe cirrhosis. IFNalpha 5 expressed in liver has the most potent antiviral activity, making it an appealing therapeutic candidate. Insulin-like Growth Factor I (IGF-I) is a potent multifunctional anabolic hormone produced by the liver, and its deficiency results in several systematic complications occurring in liver cirrhosis. The annual requirement of IGF-l per cirrhotic patient is 600 mg, and the cost per mg is $30,000. Transgenic chloroplast technology provides a novel solution to recombinant protein production because of hyper-expression capabilities and ability to fold and process eukaryotic proteins with disulfide bridges (thereby eliminating the need for expensive post-purification processing). Tobacco is an ideal choice because of its large biomass, ease of scale-up (million seeds pen plant), genetic manipulation and an impending need to explore alternative uses for this hazardous crop. Oral delivery of functional biopharmaceuticals reduces 90 percent of the production cost, eliminating the need for expensive purification. Furthermore, bio-encapsulation within plant cells offers protection against proteolytic degradation during digestion. Interferon given orally has biological activity in human and animals. Therefore, IFNalpha2, IFNalpha5 and IGF-l will be expressed as follows: a) Develop recombinant DNA vectors for enhanced expression or oral delivery b) Generate transgenic tobacco and tomato plants c) Characterize transgenic expression of proteins or fusion proteins using molecular and biochemical methods d) Purify therapeutic proteins from transgenic chloroplasts e) Characterize and compare therapeutic proteins (yield, purity, functionality) produced in yeast, E.coli or transgenic plants f) Study in vitro and in vivo (pre-clinical trials) protein bio-functionality, parenteral and oral delivery. Large scale and low cost production via transgenic chloroplasts should provide treatment to patients at an affordable cost and tobacco farmers alternate uses for this hazardous crop.